Strategies that increase homing to the bone marrow and engraftment efficacy of ex vivo expended CD34+ cells are expected to enhance their clinical utility. Here we report that nicotinamide (NAM), a form of vitamin B-3, delayed differentiation and increased engraftment efficacy of cord blood-derived human CD34+ cells cultured with cytokines. In the presence of NAM, the fraction of CD34+CD38- cells increased and the fraction of differentiated cells (CD14+, CD11b+, and CD11c+) decreased. CD34+ cells cultured with NAM displayed increased migration toward stromal cell derived factor-1 and homed to the bone marrow with higher efficacy, thus contributing to their increased engraftment efficacy, which was maintained in competitive transplants with noncultured competitor cells. NAM is a known potent inhibitor of several classes of ribosylase enzymes that require NAD for their activity, as well as sirtuin (SIRT1), class III NAD+-dependent-histone-deacetylase. We demonstrated that EX-527, a specific inhibitor of SIRT1 catalytic activity, inhibited differentiation of CD34+ cells similar to NAM, while specific inhibitors of NAD-ribosylase enzymes did not inhibit differentiation, suggesting that the NAM effect is SIRT1-specific. Our findings suggest a critical function of SIRT1 in the regulation of hematopoietic stem cell activity and imply the clinical utility of NAM for ex vivo expansion of functional CD34+ cells.